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BACKGROUND AND AIMS: NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific, HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or an NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis, and HCC development were studied by histology, flow cytometry, quantitative PCR, and RNA sequencing. The liver lipidome was characterized by lipidomics analysis, and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation sequencing. Hepatocyte-specific, HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition compared to control HuR-sufficient mice. On an NAFLD-inducing diet, hepatocyte-specific HuR deficiency resulted in exacerbated inflammation, fibrosis, and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics, and RNA immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady state, a triglyceride signature resembling that of NAFLD livers. Moreover, up-regulation of secreted phosphoprotein 1 expression mediated, at least partially, fibrosis development in hepatocyte-specific HuR deficiency on an NAFLD-inducing diet, as shown by experiments using antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis, preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically., Hepatology, 75 (4), ISSN:0270-9139, ISSN:1527-3350

In response to oncogenic signals, Alternative Splicing (AS) regulators such as SR and hnRNP proteins show altered expression levels, subnuclear distribution and/or post-translational modification status, but the link between signals and these changes remains unknown. Here, we report that a cytosolic scaffold protein, IQGAP1, performs this task in response to heat-induced signals. We show that in gastric cancer cells, a nuclear pool of IQGAP1 acts as a tethering module for a group of spliceosome components, including hnRNPM, a splicing factor critical for the response of the spliceosome to heat-shock. IQGAP1 controls hnRNPM's sumoylation, subnuclear localisation and the relevant response of the AS machinery to heat-induced stress. Genome-wide analyses reveal that IQGAP1 and hnRNPM co-regulate the AS of a cell cycle-related RNA regulon in gastric cancer cells, thus favouring the accelerated proliferation phenotype of gastric cancer cells. Overall, we reveal a missing link between stress signals and AS regulation. InfrafrontierGR/Phenotypos Infrastructure, co-funded by Greece and the European Union (European Regional Development Fund) [NSRF 2014–2020, MIS 5002135]; Hellenic Foundation for Research & Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT) [grant agreement 846 to ZE]; MR was supported by the European Research Council [ERC AdvG 670146]; European Commission Grant FP7-PEOPLE-2010-IEF [274837] to PK; Stavros Niarchos Foundation (SNF) donation to BSRC “Al. Fleming”.

Leishmaniosis is a zoonotic disease that affects millions of people especially in resource-poor settings. The development of reliable diagnostic assays that do not require dedicated equipment or highly trained personnel would improve early diagnosis and effective control. For this purpose, a combination of magnetic bead and cadmium selenite quantum dot probes was applied for the detection of Leishmania-specific surface antigens (proteins) and DNA. Both analytes are isolated from the solution using magnetic bead capture probes whereas the presence of the targeted molecules is demonstrated by quantum dot detection probes. The sensitivity and specificity of this method reached 100% based on an assessment performed on 55 cultured isolates of various microbial pathogens. The low limit of detection was 3125 ng/μl and 10(3)cells/ml for Leishmania DNA and protein, respectively. The method shows considerable potential for clinical application in human and veterinary medicine, especially in resource-poor settings.

The study aimed to investigate whether the genetic polymorphisms in the 3 ′ UTR of the caprine SLC11A1 gene are functional, and to assess the role of MAP as a regulatory parameter in gene expression. To this goal we constructed plasmids expressing the Luciferase reporter gene in transient transfections of a mouse (Balb/c) macrophage cell line (RAW264.7), incorporating those polymorphisms that our previous work indicated as more prominent in terms of SLC11A1 expression and responsiveness to MAP infection. Gene expression variation was recorded on the average of the respective measurements after exposure to Mycobacterium avium subsp. paratuberculosis (MAP) combined with microbial antigens and cytokines. In silico analysis of the region under study allowed identification of one cis-acting RNA element, five putative transcriptional regulatory elements and 85 3 ’ end microRNA binding sites. The two polymorphic regions (regions A and B) of the 3 ’ UTR of the caprine SLC11A1 gene were recognized as regulators of its activity, at transcriptional and post-transcriptional level. The GT16 polymorphism at region A, combined with the GT8 polymorphism at region B, results in up-regulation of the SLC11A1 gene. The specific genotype was also found to be more responsive to MAP exposure at a statistically significant level.

Natural infection of domestic cats by Leishmania infantum (synonym: L. chagasi) has been demonstrated in several European, Latin American, and Asian countries, and the estimated prevalence of infection, based mainly on blood PCR, ranges from 0.3% up to 60.6%. In this study we aimed to: (a) estimate the prevalence of the infection by L. infantum in clinically normal cats (group A; n = 50) and in cats with various clinical signs (group B; n = 50), living in an endemic region, by both cytological examination of four different tissues (lymph node, skin, bone marrow, and conjunctiva) and by PCR in four different tissues (blood, skin biopsies, bone marrow, and conjunctiva); (b) compare the diagnostic sensitivity of the above methods and evaluate for possible associations between their results; and (c) investigate the possible associations between infection by L. infantum and signalment, living conditions, season of sampling, and health status of the cats. The prevalence of the infection in the study population was 41% and did not differ (P = 0.839) between group A (42%) and B (40%) cats. Lymph node, skin, bone marrow and conjunctiva cytology was always negative. Therefore, the diagnosis of the infection was based only on PCR in blood, skin biopsy, bone marrow and conjunctiva, which was positive in 13%, 18.2%, 16% and 3.1% of the cats, respectively. PCR was positive in only one of the four tissues in 80.5% of the infected cats. The results differed (P = 0.014) among the four tissues and were less frequently positive in conjunctiva compared to skin biopsies and bone marrow (P = 0.007 for both comparisons), thus highlighting the need for multiple tissue PCR testing in order to minimize false-negative results. More PCR-positive cats were found when sampling was performed during the period of sandfly activity (odds ratio: 2.44; P = 0.022). Also, in group B cats, the likelihood of PCR-positivity was higher (odds ratio: 3.93; P = 0.042) among those presenting at least one systemic clinical sign that had been previously reported in cats with leishmaniosis.

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.

The aim of the present study was to assess the presence of single nucleotide polymorphisms (SNPs) in the Growth Differentiating Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) genes, in two Greek sheep breeds i.e. Chios and Karagouniki, with high and moderate litter size (LS) i.e. number of lambs born, respectively. An examination of association(s) between the polymorphisms and LS, within breeds, was also conducted. Blood samples from 92 Chios and 96 Karagouniki ewes were collected, while repeated records on LS, were also available ( n = 239 and 259, respectively). Detection of SNPs was performed on the DNA products by PCR-RLFP analysis. Data analysis included examination of allelic and genotypic differentiation between the breeds, testing for Hardy–Weinberg Equilibrium (HWE) and association analysis between polymorphisms and LS. Genetic analysis showed that the G1 and G8 mutations of the GDF9 gene were significantly over-presented only in the highly prolific breed (Chios). The B4 mutation of the BMP15 gene was significantly over-presented only in the low prolific breed (Karagouniki). Both breeds were in Hardy–Weinberg disequilibrium (HWD), possibly due to selection pressure, population sub-structure and genetic drift. Certain polymorphisms of the G1, G4 and G8 sites of the GDF9 gene in the Chios breed were found to show heterozygote advantage with no evidence of infertility for the homozygous females. A similar trend was observed in the BMP15 gene although at no statistically significant level. In the Karagouniki breed, no significant associations between the studied polymorphisms and LS were detected.

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A–D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.

Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75 ng of mycobacterial DNA diluted in a sample-volume of 10 mu l. In order to obtain an indication of the methods performance on clinical samples we applied the optimized assay to the detection of Mycobacterium avium subsp. paratuberculosis DNA in faeces, in comparison with real-time PCR. The concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%. The proposed method could be used as a highly specific and sensitive screening tool for the detection of mycobacteria directly from clinical samples in a very simple manner, without the need of high-cost dedicated equipment. The technology described here, may develop into a platform that could accommodate detection of many bacterial species and could be easily adapted for high throughput and expedite screening of samples. (C) 2009 Elsevier B.V. All rights reserved.

Paratuberculosis, is a disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects mainly ruminants. MAP can disseminate from the intestine to the genital tract. Therefore infection has been associated with decreased fertility but not directly with abortion in goats. The aim of this study was to obtain the evidence required to exploit the potential association between MAP-infection and abortion. For this purpose we have focused on three caprine herds geographically unrelated and keeping records of paratuberculosis and abortion of unspecified etiology. We collected in total 178 serum, milk, and fecal samples from 63, 2-4 years old, female goats. This material was processed for cultiva- tion, ELISA, and Real Time PCR (RT-PCR). No statistically significant association was recorded between abortion- record and RT-PCR positivity to MAP of fecal or milk samples. However ELISA - positivity was linked at a statistically significant level (p = 0.0275) with abortion. Goats that reacted positively by ELISA were 3.24 times more likely to abort than those with no detectable antibody titer. This study provides the first indications of the potential association of MAP- infection in goats and abortion-record, and suggests that a more extensive investigation should preferably rely on serol- ogy.

Highlights • IFAT for anti-Leishmania IgG was positive in 10/100 cats. • ELISA for anti-Leishmania IgG was positive in 1/100 cats. • IFAT for anti-Leishmania IgM was positive in 1/100 cats. • The results of IFAT and ELISA for anti-Leishmania IgG disagreed (P = 0.039). • IFAT or ELISA for anti-Leishmania IgG disagreed with the results of PCR., Graphical Abstract, Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P

Leishmaniosis is a zoonose caused by protozoans of the genus Leishmania. The need for accurate diagnostic investigation of cases of leishmaniosis has rendered today the use of molecular biology techniques broadly applicable. However, the reliable application of these methods requires highly-specialised personnel, dedicated equipment and space. The aim of this study was the design and construction of functionalized gold nanoparticles (AuNPs) that would be incorporated into an easily applicable DNA detection methodology for the identification of Leishmania spp. in clinical samples. AuNPs 20nm in diameter were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. In the absence of complimentary DNA, AuNPs-probes precipitate under acid environment causing a change of color from red to purple, which can be detected by visual observation. In the presence of target DNA the color of the solution remains red. The specific methodology was applied to positive and negative control samples and whole blood collected from dogs with suspected canine leishmaniosis. The method's minimum detection limit was defined to 11.5ng of target DNA per μl of sample. Repeatability and reproducibility were 100%. Relative sensitivity and specificity referenced to PCR were calculated to 92% and 100% regarding collectively control and clinical samples. The proposed approach can be considered an appealing diagnostic solution especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not top priority.

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.

Johne's disease or paratuberculosis is a chronic, progressive intestinal disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). One of the genes that have been targeted with regard to resistance or sensitivity to paratuberculosis is the SLC11A1 (solute carrier family 11 member A1). Here we extend our previous work to the sequence and structure analysis of the caprine SLC11A1 gene and we assess the functional impact of the most frequent polymorphisms of the 3' UTR region of the SLC11A1 gene to its expression in goat macrophages exposed in vitro to MAP. The role of these polymorphisms in primary immune response is also investigated with connection to gene expression of two interleukins (IL), one of which pro (IL-1a), and the other anti-inflammatory (IL-10). In order to assess gene response, quantitative detection of the SLC11A1, IL-10 and IL1a mRNA was performed by real time PCR before, and at 1, 3 and 24h after exposure of primary cultures of peripheral blood monocyte-derived macrophages to MAP, collected from 54 goats of the Greek native goat breed. Sequence analysis of the 3' UTR end of the caprine SLC11A1 gene determined its full length to be 522 bases. Structure analysis confirmed the presence of two microsatellites consisted of a variable number of guanine-thymine repeats (regions A and B). The homozygous B7 genotype [B(GTn)7/7] was associated at a statistically significant level with increased expression of the SLC11A1 and IL-1α genes indicating increased in vitro responsiveness and therefore resistance of mononuclear derived macrophages to MAP infection.

Here we present the development of a specific DNA detection method using fluorescent semiconductor quantum dots (QDs) and magnetic beads (MBs) for fast detection of Mycobacterium spp., dispensing with the need for DNA amplification. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary mycobacterial target DNA through a sandwich hybridization reaction. Cadmium selenite QDs conjugated with streptavidin and species-specific probes were used to produce a fluorescent signal. MBs conjugated with streptavidin and a genus-specific probe were used to isolate and concentrate the DNA targets. The application of the proposed method to isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined as 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance with clinical samples, we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage specimens from patients with tuberculosis and Mycobacterium avium subsp. paratuberculosis in DNA isolated from feces and paraffin-embedded tissues in comparison with culture, Ziehl-Neelsen staining, and real-time PCR. The concordance of these methods compared to the proposed method with regard to positive and negative samples varied between 53.84% and 87.23% and between 84.61% and 100%, respectively. The overall accuracy of the QD assay compared to real-time PCR was 70 to 90% depending on the type of clinical material. The proposed diagnostic assay offers a simple, rapid, specific, and cost-effective method for direct detection and identification of mycobacterial DNA in clinical samples.